The failure of Ad5 based HIV-1 vaccine in humans that is designed to elicit primarily antiviral T cells strongly suggests the need to develop novel vaccine approaches that generate high levels of anti-viral T cells with improved function as well as protective Ab. The goal of this project is to adjuvant the cellular and humoral immunity elicited by our DNA/MVA vaccine that has just entered phase II testing in humans in US. Specifically, we propose to target the CD40 pathway on dendritic cells (DC) and B cells using CD40L. Stimulation of CD40 on DC results in production of IFNg and IL-12 that are critical for generation of highly functional antiviral CDS response. Activation of B cells through CD40 is necessary for germinal center formation where affinitv maturation of B cells occurs leading to generation of high avidity Ab. Here, we will express CD40L on the surface of a SIV virus-like particle (CD40L-VLP). These CD40L-VLPs are potent activators of DC and B cells in vitro. In addition, they can be targeted to DC (through interaction between gpl20 on VLP and CD4 on DC) and allow presentation of Env in its native form that is critical for eliciting broadly cross-reactive neutralizing Ab. In specific aim 1. we will test the potential of CD40L-adiuvanted DNA/MVA SIV vaccine to enhance control of a pathogenic SIV challenge. In addition, we will test whether delivering DNA by electroporation enhances the immunogenicity of the adluvanted and non-adiuvanted DN/VMVA vaccines. Recent studies from Dr. Rafi Ahmed's lab (PI of project 2) demonstrated that mTOR regulates memory T cell development and inhibition of this pathway following infection or vaccination using rapamycin enhances the magnitude and functional quality of antigen-specific CDS T cells. Rapamycin has also been shown to down regulate expression of CCR5 on CD4 T cells that results in marked reduction of HIV replication. This could be an added advantage for HIV vaccines, because the vaccine-elicited CCR5' virus-specific CD4 T cells may not be infected by the virus. Essentially we may be reducing the freguency of potential virus target cells while preserving the much-needed CD4 T cell helD following infection. In specific aim 2, we will test the synergy between inhibiting mTOR and activating CD40 pathways for adjuvanting the immunogenicity and efficacy of DNA/MVA vaccines.